Thiol-Ene Click Hydrogels for in situ Cell Expansion and Differentiation

描述（由申请人提供）：水凝胶是亲水性聚合物基质，可以支持细胞和组织的三维生长。经过精心设计，水凝胶不仅可以作为研究细胞行为的 3D 平台，还可以作为培养和分化干/祖细胞的支架。我们的目标是开发一种多功能水凝胶系统，该系统最初支持细胞增殖，但在后期可以“转换”到促进细胞/组织分化为特定细胞类型的微环境中。我们的中心假设是，细胞扩增和分化可以在结合动态生物物理和生化线索的单一水凝胶基质中实现。我们将通过使用多种细胞相容性硫醇-烯“点击”反应开发多功能水凝胶系统来测试这一假设。在初始阶段，我们将设计局部可降解的硫醇烯凝胶来促进PANC-1细胞（胰腺导管上皮细胞系）的增殖（目标1）。我们将结合基质金属蛋白酶 2 (MMP-2) 特异性肽底物作为水凝胶交联剂。这允许凝胶通过 PANC-1 细胞分泌的 MMP-2 进行局部降解（从而为细胞增殖创造额外的空间）。通过调节水凝胶中的细胞-ECM和细胞-细胞相互作用，以及为封装细胞提供可扩散的可溶性生长因子，将促进封装细胞的增殖。接下来，我们将把充满细胞的水凝胶从“促增殖”微环境“切换”为“促分化”微环境（目​​标 2a）。我们将通过进行第二次硫醇-烯点击反应来实现这一目标，以在充满细胞的水凝胶内引入促分化线索，从而促进胰岛样胰岛素分泌细胞簇的形成。分化过程将是使用无血清培养基、基于亲和力的碱性成纤维细胞生长因子 (bFGF) 募集以及及时缀合胰高血糖素样肽 1 (GLP-1) 的综合结果。可以从易蚀凝胶中回收分化的细胞簇（由于特定的酶活性），以进行进一步的表征和生物/临床应用（目标 2b）。由于强烈的细胞间相互作用，分化的簇预计会形成紧密的聚集体。总之，我们的策略促进了具有自然细胞间相互作用和正常胰岛素分泌特征的胰岛样细胞簇的形成。

DESCRIPTION (provided by applicant): Hydrogels are hydrophilic polymeric matrices that can support cell and tissue growth in three-dimension. With careful design, hydrogels serve not only as a 3D platform for studying cell behaviors, but also as scaffolds for culturing and differentiating stem/progenitor cells. We aim to develop a multifunctional hydrogel system that initially supports cell proliferation, but at a later stage can be "switched" into a microenvironment that promotes cell/tissue differentiation into a specific cell type. Our central hypothesis is that cell expansion and differentiation can be achieved in a single hydrogel matrix incorporated with dynamic biophysical and biochemical cues. We will test this hypothesis by developing a versatile hydrogel system using multiple cytocompatible thiol-ene "click" reactions. In the initial stage, we will design locally degradable thiol-ene gels to promote proliferation of PANC-1 cells (a pancreatic ductal epithelial cell line) (Aim 1). We will incorporate matrix metalloproteinase 2 (MMP-2) specific peptide substrates as hydrogel crosslinkers. This allows the gels to degrade locally (thus creating additional space for cell proliferation) by MMP-2 secreted from PANC-1 cells. The proliferation of encapsulated cells will be promoted by tuning cell-ECM and cell-cell interactions in hydrogels, as well as providing the encapsulated cells with diffusible soluble growth factors. Next, we will "switch" the cell-laden hydrogels from a "pro-proliferation" to a "pro-differentiation" microenvironment (Aim 2a). We will achieve this by performing a second thiol-ene click reaction to introduce pro-differentiation cues within the cell-laden hydrogels, thus promoting the formation of islet-like, insulin secreting cell clusters. The differentiation process will be a combinatorial result of using serum-free culture media, affinity-based recruitment of basic fibroblast growth factor (bFGF), and timely conjugation of glucagon-like peptide 1 (GLP-1). The differentiated cell clusters can be retrieved from the erodible gels (due to specific enzyme activity) for further characterization and biological/clinical applications (Aim 2b). The differentiated clusters are expected to form tight aggregates due to strong cell-cell interactions. Together, our strategies facilitate the formation of islet-like cell clusters with natural cell-cell interactions and normal insulin secretion profiles.